Cyclosporine (CsA) is a novel immunosuppressive agent which appears to affect predoninantly T-lymphocyte dependent responses. Despite its increasing clinical usage, the specific mechanism of action of CsA is unknown. The availability of a dansylated derivative of CsA, which retains its native immunosuppressive activity but which is fluorescent, offers new and unique approaches to determine the mechanism of action of CsA. In preliminary studies, using the fluorescent-activated cell sorter (FACS), both peripheral blood T-lymphocytes, and T-cell clones have been shown to have differential staining with dansylated CsA derivatives. T-cell clones will be subjected to functional analysis to determine if diffential staining may represent functionally different cells among the OKT4 and OKT8 subsets of lymphocytes. T-cell clones will also be analyzed for Cyclosporine sensitivity resistance and correlataed with staining characteristics. Competitive inhibition studies will be performed between the dansylated Cyclosporine derivatives, and other both active and inactive derivatives of Cyclosporine. Competetive inhibition studies will also be done between the dansylated probes and reagents with known or purported receptors on T-lymphocytes including the OK monoclonal antibodies, PHA, PMA, ConA, antigen and HLA antibodies. From these competetive inhibition studies binding specificity of Cyclosporine as well as binding coefficients and dissociation constants will be derived. It may indicate if a unique receptor exists on the T-lymphocyte cell membrane for Cyclosporine. The availability of populations of T-cell clones in our laboratory, derived from both the OKT4 and OKT8 pupulations, which show differential staining characteristics with the dansylated derivatives of CsA will allow for examination of a homogeneous pupulation of T-cells. In addition, fluorescent microscopic techniques will be utilized as an adjunct to the above studies as well as a tool to further examine the significance of differential membrane and cytoplasmic binding of CsA among T-lymphocyte subsets.